Alejandra Vitale

Effects of a Gonadotropin-Releasing Hormone Agonist on Rat Ovarian Follicle Apoptosis: Regulation by Epidermal Growth Factor and the Expression of Bcl-2-Related Genes

Abstract The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3′ end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.

Gonadotropin-Releasing Hormone Antagonist Antide Inhibits Apoptosis of Preovulatory Follicle Cells in Rat Ovary

Abstract Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.

Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct

Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.

Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats.

Abstract The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor β (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. 3H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 μg/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.

PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.

Inhibin A and B levels in follicular fluid of patients undergoing assisted reproduction: correlation with hormone levels and pregnancy ☆

Over the past several years, attention has been focused on the possible autocrine and/or paracrine role of these dimers in the regulation of folliculogenesis in humans. Follicular fluid, obtained from patients undergoing assisted reproduction techniques (ART), can be used to investigate the regulatory ovarian factors of oocyte competence. Hall et al. (3) proposed that inhibin A and B may be considered as peripheral markers of ovarian function in patients who are undergoing assisted reproduction techniques. However, there are no studies that describe the correlation between the levels of inhibin A and B and the concentration of gonadotropins or steroids in follicular fluid obtained the day of oocyte retrieval. The aim of this study was to determine the levels of inhibin A and B in the follicular fluid obtained from patients undergoing assisted reproduction techniques and to correlate these levels with 1) the age of the patients, 2) the number of oocytes recovered, 3) the concentrations of follicular FSH, hCG, estradiol (E 2 ), and progesterone, and 4) the pregnancy outcome. We made a retrospective analysis of 23 patients who were undergoing IVF. Follicular fluid was aspirated 36 hours after hCG administration, and the oocytes were immediately recovered. For each patient, the cells and fluid from the follicles at the same stage of maturation ( .16 mm diameter, grade of oocyte maturation: 4, n 5 4 ‐17) were pooled. The follicular fluid was centrifuged and the supernatant (the follicular fluid) was frozen at 220°C until the assay. The concentration of inhibin A and B in the follicular fluid was determined with the use of a two-site enzyme-linked immunosorbent assay (ELISA) that was specific for each peptide (4). FSH, hCG, progesterone, and E2 were measured by radioimmune assay. The correlation between the levels of inhibin A, inhibin B, steroids, and gonadotropins in the follicular fluid was determined.